Steroid receptors mediate their biological activities by altering the expression rate of a subset of genes in target tissues. Current concepts of gene regulation emphasize the role of protein-protein interactions between transcriptional activators (such as the steroid receptors) and components of the basal transcription machinery, as the dominant mechanism in differential promoter activity. During the course of experiments designed to address gene activation by the progesterone and glucocorticoid receptors, we discovered that a target promoter that is normally responsive to progesterone activation in transient transfection systems can be completely refractory to stimulation when organized in stable, replicated chromatin. These observations led to the suggestion that receptor access to target promoters can be functionally modified by the nucleoprotein status of the regulatory element, and open the general possibility that chromatin structure serves as an additional layer of regulation for these biologically important molecules. To address these issues critically, we have evolved a new experimental approach that permits the direct comparison of transactivator interactions with stable, replicated chromatin, in contrast to transiently introduced, disorganized templates. Using fluorescence activated cell sorting, we have purified the population of cells that acquire transiently introduced DNA, permitting an analysis of the interaction of trans-regulators with transfected reporter constructions, as opposed to chromosomal genes. Using this new technique, we confirmed that a target promoter in stable chromatin can be completely resistant to hormone induction, although the soluble components for transactivation are present and functional.